The Populus holobiont: dissecting the effects of plant niches and genotype on the microbiome.

BACKGROUND: Microorganisms serve important functions within numerous eukaryotic host organisms. An understanding of the variation in the plant niche-level microbiome, from rhizosphere soils to plant canopies, is imperative to gain a better understanding of how both the structural and functional processes of microbiomes impact the health of the overall plant holobiome. Using Populus trees as a model ecosystem, we characterized the archaeal/bacterial and fungal microbiome across 30 different tissue-level niches within replicated Populus deltoides and hybrid Populus trichocarpa × deltoides individuals using 16S and ITS2 rRNA gene analyses. RESULTS: Our analyses indicate that archaeal/bacterial and fungal microbiomes varied primarily across broader plant habitat classes (leaves, stems, roots, soils) regardless of plant genotype, except for fungal communities within leaf niches, which were greatly impacted by the host genotype. Differences between tree genotypes are evident in the elevated presence of two potential fungal pathogens, Marssonina brunnea and Septoria sp., on hybrid P. trichocarpa × deltoides trees which may in turn be contributing to divergence in overall microbiome composition. Archaeal/bacterial diversity increased from leaves, to stem, to root, and to soil habitats, whereas fungal diversity was the greatest in stems and soils. CONCLUSIONS: This study provides a holistic understanding of microbiome structure within a bioenergy relevant plant host, one of the most complete niche-level analyses of any plant. As such, it constitutes a detailed atlas or map for further hypothesis testing on the significance of individual microbial taxa within specific niches and habitats of Populus and a baseline for comparisons to other plant species.

Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens.

Endosymbiosis of bacteria by eukaryotes is a defining feature of cellular evolution. In addition to well-known bacterial origins for mitochondria and chloroplasts, multiple origins of bacterial endosymbiosis are known within the cells of diverse animals, plants and fungi. Early-diverging lineages of terrestrial fungi harbor endosymbiotic bacteria belonging to the Burkholderiaceae. We sequenced the metagenome of the soil-inhabiting fungus Mortierella elongata and assembled the complete circular chromosome of its endosymbiont, Mycoavidus cysteinexigens, which we place within a lineage of endofungal symbionts that are sister clade to Burkholderia. The genome of M. elongata strain AG77 features a core set of primary metabolic pathways for degradation of simple carbohydrates and lipid biosynthesis, while the M. cysteinexigens (AG77) genome is reduced in size and function. Experiments using antibiotics to cure the endobacterium from the host demonstrate that the fungal host metabolism is highly modulated by presence/absence of M. cysteinexigens. Independent comparative phylogenomic analyses of fungal and bacterial genomes are consistent with an ancient origin for M. elongata - M. cysteinexigens symbiosis, most likely over 350 million years ago and concomitant with the terrestrialization of Earth and diversification of land fungi and plants.

A 34K SNP genotyping array for Populus trichocarpa: design, application to the study of natural populations and transferability to other Populus species.

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost-effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre-ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids.

Revisiting the sequencing of the first tree genome: Populus trichocarpa.

Ten years ago, it was announced that the Joint Genome Institute with funds provided by the Department of Energy, Office of Science, Biological and Environmental Research would sequence the black cottonwood (Populus trichocarpa Torr. & Gray) genome. This landmark decision was the culmination of work by the forest science community to develop Populus as a model system. Since its public release in late 2006, the availability of the Populus genome has spawned research in plant biology, morphology, genetics and ecology. Here we address how the tree physiologist has used this resource. More specifically, we revisit our earlier contention that the rewards of sequencing the Populus genome would depend on how quickly scientists working with woody perennials could adopt molecular approaches to investigate the mechanistic underpinnings of basic physiological processes. Several examples illustrate the integration of functional and comparative genomics into the forest sciences, especially in areas that target improved understanding of the developmental differences between woody perennials and herbaceous annuals (e.g., phase transitions). Sequencing the Populus genome and the availability of genetic and genomic resources has also been instrumental in identifying candidate genes that underlie physiological and morphological traits of interest. Genome-enabled research has advanced our understanding of how phenotype and genotype are related and provided insights into the genetic mechanisms whereby woody perennials adapt to environmental stress. In the future, we anticipate that low-cost, high-throughput sequencing will continue to facilitate research in tree physiology and enhance our understanding at scales of individual organisms and populations. A challenge remains, however, as to how genomic resources, including the Populus genome, can be used to understand ecosystem function. Although examples are limited, progress in this area is encouraging and will undoubtedly improve as future research targets the many unique aspects of Populus as a keystone species in terrestrial ecosystems.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Cytogenetic analysis of Populus trichocarpa--ribosomal DNA, telomere repeat sequence, and marker-selected BACs.

The 18S-28S rDNA and 5S rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 18S-28S rDNA sites and one 5S rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones selected from 2 linkage groups based on genome sequence assembly (LG-I and LG-VI) were localized on 2 chromosomes, as expected. BACs from LG-I hybridized to the longest chromosome in the complement. All BAC positions were found to be concordant with sequence assembly positions. BAC-FISH will be useful for delineating each of the Populus trichocarpa chromosomes and improving the sequence assembly of this model angiosperm tree species.

Microsatellite primer resource for Populus developed from the mapped sequence scaffolds of the Nisqually-1 genome.

In this study, 148 428 simple sequence repeat (SSR) primer pairs were designed from the unambiguously mapped sequence scaffolds of the Nisqually-1 genome. The physical position of the priming sites were identified along each of the 19 Populus chromosomes, and it was specified whether the priming sequences belong to intronic, intergenic, exonic or UTR regions. A subset of 150 SSR loci were amplified and a high amplification success rate (72%) was obtained in P. tremuloides, which belongs to a divergent subgenus of Populus relative to Nisqually-1. PCR reactions showed that the amplification success rate of exonic primer pairs was much higher than that of the intronic/intergenic primer pairs. Applying ANOVA and regression analyses to the flanking sequences of microsatellites, the repeat lengths, the GC contents of the repeats, the repeat motif numbers, the repeat motif length and the base composition of the repeat motif, it was determined that only the base composition of the repeat motif and the repeat motif length significantly affect the microsatellite variability in P. tremuloides samples. The SSR primer resource developed in this study provides a database for selecting highly transferable SSR markers with known physical position in the Populus genome and provides a comprehensive genetic tool to extend the genome sequence of Nisqually-1 to genetic studies in different Populus species.

A genetic linkage map for the ectomycorrhizal fungus Laccaria bicolor and its alignment to the whole-genome sequence assemblies.

A genetic linkage map for the ectomycorrhizal basidiomycete Laccaria bicolor was constructed from 45 sib-homokaryotic haploid mycelial lines derived from the parental S238N strain progeny. For map construction, 294 simple sequence repeats (SSRs), single-nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) markers were employed to identify and assay loci that segregated in backcross configuration. Using SNP, RAPD and SSR sequences, the L. bicolor whole-genome sequence (WGS) assemblies were aligned onto the linkage groups. A total of 37.36 Mbp of the assembled sequences was aligned to 13 linkage groups. Most mapped genetic markers used in alignment were colinear with the sequence assemblies, indicating that both the genetic map and sequence assemblies achieved high fidelity. The resulting matrix of recombination rates between all pairs of loci was used to construct an integrated linkage map using JoinMap. The final map consisted of 13 linkage groups spanning 812 centiMorgans (cM) at an average distance of 2.76 cM between markers (range 1.9-17 cM). The WGS and the present linkage map represent an initial step towards the identification and cloning of quantitative trait loci associated with development and functioning of the ectomycorrhizal symbiosis.

The genome of black cottonwood, Populus trichocarpa (Torr. & Gray).

We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

Large-scale heterospecific segregation distortion in Populus revealed by a dense genetic map.

We report the most complete genetic map to have been constructed for the genus Populus. This map includes 544 markers mapped onto 19 linkage groups, equivalent to the Populus chromosome number, with all markers displaying internally consistent linkage patterns. We estimate the genome length to be between 2,300 and 2,500 cM, based both on the observed number of crossovers in the maternal haplotypes, as well as the total observed map length. Genome coverage was estimated to be greater than 99.9% at 20 cM per marker. We did not detect obvious recombination repression in the maternal tree (a hybrid of Populus trichocarpa Hooker x P. deltoides Marsh.) compared to the paternal tree (pure P. deltoides). Finally, most markers exhibiting segregation distortion were derived from the donor parent in this backcross, and generally occurred in large contiguous blocks on two linkage groups. We hypothesize that divergent selection has occurred on chromosomal scales among the parental species used to create this pedigree, and explore the evolutionary implications of this observation. This genetic linkage map provides the most comprehensive view of the Populus genome reported to date and will prove invaluable for future inquiries into the structural and functional genomics, evolutionary biology, and genetic improvement of this ecologically important model species.

Poplar genomics is getting popular: the impact of the poplar genome project on tree research.

Trees, due to their long life-span, have characteristics that distinguish them from annual, herbaceous plants. It is likely that many of these properties are based on a tree-specific genetic foundation. The U.S. Department of Energy initiated a genome-sequencing project for Populus, a model perennial plant. Through international collaboration and input to the sequencing effort, the annotated whole genome sequence of Populus trichocarpa will be released to the public in early 2004. This genomic resource will, for the first time, allow comparison between a perennial and an annual plant on a whole genome basis and therefore provide clues for molecular research on tree-specific questions like dormancy, development of a secondary cambium, juvenile-mature phase change, or long-term host-pest interactions. The approximately 520 Mbp of annotated genomic sequence will complement and expand the knowledge provided so far by the 125,000 ESTs from poplar that are available in public databases. This article introduces the international poplar research programmes and points out the significance of the poplar genome project for plant research.

Genetic and physical mapping of Melampsora rust resistance genes in Populus and characterization of linkage disequilibrium and flanking genomic sequence.

• In an attempt to elucidate the molecular mechanisms of Melampsora rust resistance in Populus trichocarpa, we have mapped two resistance loci, MXC3 and MER, and intensively characterized the flanking genomic sequence for the MXC3 locus and the level of linkage disequilibrium (LD) in natural populations. • We used an interspecific backcross pedigree and a genetic map that was highly saturated with AFLP and SSR markers, and assembled shotgun-sequence data in the region containing markers linked to MXC3. • The two loci were mapped to different linkage groups. Linkage disequilibrium for MXC3 was confined to two closely linked regions spanning 34 and 16 kb, respectively. The MXC3 region also contained six disease-resistance candidate genes. • The MER and MXC3 loci are clearly distinct, and may have different mechanisms of resistance, as different classes of putative resistance genes were present near each locus. The suppressed recombination previously observed in the MXC3 region was possibly caused by extensive hemizygous rearrangements confined to the original parent tree. The relatively low observed LD may facilitate association studies using candidate genes for rust resistance, but will probably inhibit marker-aided selection.

Highly variable SSR markers in Douglas-fir: Mendelian inheritance and map locations.

Twenty-two highly variable SSR markers were developed in Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco] from five SSR-enriched genomic libraries. Fifteen PCR primer pairs amplified a single codominant locus, while seven primer pairs occasionally amplified two loci. The Mendelian inheritance of all 22 SSRs was confirmed via segregation analyses in several Douglas-fir families. The mean observed heterozygosity and the mean number of alleles per locus were 0.855 (SE=0.020) and 23 (SE=1.6), respectively. Twenty markers were used in genetic linkage analysis and mapped to ten known linkage groups. Because of their high polymorphism and unambiguous phenotypes, 15 single-locus markers were selected as the most suitable for DNA fingerprinting and parentage analysis. Only three SSRs were sufficient to achieve an average probability of exclusion from paternity of 0.998 in a Douglas-fir seed orchard block consisting of 59 parents.

The development of two flanking SCAR markers linked to a sex determination locus in Salix viminalis L.

Most studies of sex determination systems in plants involve dioecious annuals that have known sex chromosomes. Despite the absence of such structures in the majority of dioecious plants, gender seems to be under relatively strict genetic control in some species. Genetic markers linked to a female sex-determination locus in Salix viminalis L. have been discovered through bulked segregant analysis of three full-sib families using approximately 1,000 arbitrary primers. Two RAPD markers that were present in the common female parent as well as in predominantly female progeny of these families were subsequently sequenced and converted to sequence characterized amplified region (SCAR) markers. The two SCAR markers are correlated with gender in the three full-sib families and are present in 96.4% of the female progeny and 2.2% of the males, providing evidence of linkage to a putative female-specific locus associated with gender determination in S. viminalis. Estimates of recombination suggest that the two markers are flanking a putative sex determination locus, SDL-II, in certain families of S. viminalis.

Genomics and the tree physiologist.

During the last decade, a strong case has been made for viewing trees as model systems in plant biology. More recently, forest biologists have argued for the sequencing of the genome of a forest tree. Now, the United States Department of Energy has announced plans for sequencing the genome of the Populus trichocarpa clone, "Nisqually-1." Thus, forest biology is poised to enter an exciting period of scientific discovery. Embracing new technology and new research paradigms, however, is never easy. It is, therefore, timely to ask how tree physiologists will take advantage of the Populus genome data. We contend that the most attractive opportunities will arise through genome-wide research designed to: (1). examine the differences between trees and herbaceous annuals; (2). explore questions relating to the temporal and spatial scales that characterize the life histories and growth of trees; and (3). investigate cause-and-effect relationships that are intractable to conventional research methodologies. To highlight the potential applications of genomics in tree physiology, we briefly discuss each of these approaches.

Identification of QTLs influencing wood property traits in loblolly pine ( Pinus taeda L.). II. Chemical wood properties.

Chemical wood property traits were analyzed for the presence of quantitative trait loci (QTLs) in a three-generation outbred pedigree of loblolly pine ( Pinus taeda L.). These traits were assayed using pyrolysis molecular beam mass spectrometry and include mass spectrum peak intensities associated with carbohydrates, alpha-cellulose and hemicellulose sugars, and lignin. Models for projection to latent structures (PLS) were used to also estimate the chemical composition of cell walls (i.e., alpha-cellulose, galactan and lignin) from mass spectrum data using multivariate regression. Both earlywood and latewood fractions from the fifth annual ring were analyzed for each trait. An interval mapping approach designed for an outbred pedigree was used to estimate the number of QTLs, the magnitude of QTL effects, and their genomic position. Eight unique QTLs influencing cell wall chemistry were detected from multiple peak intensities and/or PLS estimates using the one- and two-QTL models. Significant differences in chemical contents were observed among the populations from North Carolina vs Oklahoma, and results from QTLxenvironment analyses suggest that QTLs interact with environmental location. QTLs should be verified in larger experiments and in different genetic and environmental backgrounds. QTL mapping will help towards eventually identifying genes having a major effect on chemical wood properties.